Figure 5

Coalescence of lipid rafts isolated, detergent-free, from T cell - APC co-cultures. YH16.33 cells were co-cultured with A20 cells in the absence (A & B) or presence (C & D) of 1 mg/ml chicken ovalbumin (specific antigen) for 18-24 hours and lipid rafts were isolated using the detergent-free density gradient method. Detergent-free lipid rafts in fraction 5 from YH16.33 + A20 (A), YH16.33 + A20 + chicken Ovalbumin (C) or A20 alone (E) were captured with anti-Thy-1 on formvar coated nickel grids and detected with biotinylated anti-Ly-6A.2 followed by anti-biotin conjugated to 10 nm colloidal gold and visualized with a Hitachi H-7600 transmission electron microscope. The average Feret's diameter of lipid rafts collected from fractions 5 of YH16.33 and A20 co-cultures either in the absence (B) or the presence (D) is shown. Error bars show average size (nm) +/- standard deviation. Lipid rafts from each group (T cells alone, T cells + APCs, T cells + APCs + antigen) were sized using NIH ImageJ software and their size distribution shown in nanometers is shown (F). Each micrograph was at a 40,000 × magnification. The micrograph shown are representative of at least three sets of experiments and the quantitative data is derived from all the experiments (n = 2251 lipid rafts for YH16.33+A20 and n = 2071 lipid rafts for YH16.33+A20+antigen groups).